Process for producing citric acid isocitric acid and microbial cells by fermentation

ABSTRACT

Processes for producing citric acid together with microbial cells as well as producing increased yields of citric acid and/or isocitric acid by fermentation are described. Hydrocarbonassimilating microorganisms are employed in media containing hydrocarbons as the main carbon source. The addition of alcohols, such as C1-C20 saturated alcohols, to the medium permits the accumulation of citric acid together with the obtained microbial cells. The addition of ferrocyanides, such as potassium or sodium ferrocyanide, to the medium enhances the yield of isocitric acid, while the addition of monofluoroacetic acid or a salt thereof to the medium enhances the yield of citric acid.

United States Patent [1 1 Kimura et al.

[ PROCESS FOR PRODUCING CITRIC ACID ISOCITRIC ACID AND MICROBIAL CELLSBY FERMENTATION [7 5] Inventors: Kazuo Kimura; Toru Nakanishi,

both of Tokyo, Japan [73] Assignee: Kyowa Hakko Kogyo Co., Ltd., Tokyo,Japan 22 Filed: Sept. 24, 1970 21 Appl. No.: 75,240

[30] Foreign Application Priority Data Oct. 9, i969 Japan 44/80333 Nov.21, 1969 Japan 44/92925 [52] US. Cl. 195/28 R, 195/47 [51] Int. Cl. Cl2b1/00 [58] Field of Search 195/28, 36, 37, 47, 195/49, 114

[56] References Cited UNITED STATES PATENTS 3,652,396 3/1972 Tanaka eta1. 195/28 R Nov. 20, 1973 3,563,857 2/1971 Oki et al. 195/49 FOREIGNPATENTS OR APPLICATIONS 1,571,551 6/1969 France 2,005,848 8/1970 GermanyPrimary ExaminerA. Louis Monacell Assistant Examiner-R. B. PenlandAttorney-Craig, Antonelli, Stewart & Hill [57 ABSTRACT Processes forproducing citric acid together with microbial cells as well as producingincreased yields of citric acid and/or isocitric acid by fermentationare described. Hydrocarbon-assimilating microorganisms are employed inmedia containing hydrocarbons as the main carbon source. The addition ofalcohols, such as C -C saturated alcohols, to the medium permits theaccumulation of citric acid together with the obtained microbial cells.The addition of ferrocyanides, such as potassium or sodium ferrocyanide,to the medium enhances the yield of isocitric acid, while the additionof monofluoroacetic acid or a salt thereof to the medium enhances theyield of citric acid.

14 Claims, No Drawings PROCESS FOR PRODUCING CITRIC ACID ISOCITRIC ACIDAND MICROBIAL CELLS BY FERMENTATION This invention relates to a processfor producing citric acid and isocitric acid, as well as microbialcells. More particularly, it relates to a process for the production ofcitric acid, isocitric acid and microorganism cell bodies byfermentation. Even more particularly, the invention relates to saidfermentation process using hydrocarbon-assimilating microorganisms.

Attempts to obtain various microorganism products using hydrocarbons asthe main carbon source have received a great amount of attentionrecently, and there have been numerous reports in the literatureregarding the production of microbial cells, including yeast cellbodies, amino acids, nucleic acids, organic acids, sugars, enzymes,vitamins, etc.

Insofar as the production of citric acid is concerned, among the organicacids, processes based upon the use of bacteria, the use of molds andthe use of yeasts have previously been developed in the art. However,these processes are not satisfactory for producing citric acid becausethe production yields are low, the culturing times are prolonged and theproduct is sometimes obtained as a mixture with isocitric acid, which isdifficult to separate from citric acid.

With respect to the production of microorganism cell bodies, improvedmethods for producing high yields of microbial cells from hydrocarbonshave been developed with a consequent reduction in the production cost.However, since most of the microbial cells are utilized as animal feed,it is necessary and desirable to further lower the cost of production.On the other hand, the formation of microbial cells from hydrocarbons iscarried out through complicated reactions in the living body. It hasbeen found to be very useful to recover and utilize beneficialsubstances in the metabolization step in these reactions in order toeffectively utilize raw material resources. However, in such a case, theprincipal object is to solely produce cell bodies and, therefore, it isnatural that only a small amount of by-products is formed in the cultureliquor. Furthermore, as the production of the microbial cells is carriedout on a mass production scale, it isnecessary to treat a very largeamount of broth. Accordingly, the recovery of the byproducts from theculture liquor is restricted only to the recovery of readily recoverableproducts. In this respect, citric acid has a low solubility and, if itis present in the calcium salt form, it is substantially insoluble inhot water. Therefore, it is very easy to recover citric acid from suchbroths.

The processes for producing citric acid mentioned above, involving theuse of bacteria, the use of molds or the use of yeasts, are directedonly to the production of citric acid, wherein the citric acid is formedby controlling the growth of the microbial cells in a suitable manner,for example, by the addition of a metabolisminhibiting agent,restriction of the amount of nitrogen or control of some other nutrientsource, and, hence, bringing about some deviation in the metabolism. Onthe other hand, when microbial cells are produced from hydrocarbons inaccordance with the standard procedures, sufficient and necessarynutrient sources for the formation of the microbial cells are employed,with the result that only a very small amount of the metabolismintermediate substances is accumulated in the culture liquor and no orsubstantially no citric acid is detected at all.

Using these criteria, the present inventors have sought ways ofaccumulating and producing citric acid and isocitric acid byfermentation, while also producing significant amounts of microbialcells. In doing these studies, an improved process for producing citricacid and isocitric acid by fermentation has also been developed.

Accordingly, one of the objects of the present invention is to providean improved process for the production of citric acid and isocitric acidwhich overcomes the disadvantages and deficiencies of the prior artmethods.

Another object of the present invention is to provide a process forproducing considerable amounts of citric acid together with microbialcells by fermentation which may be carried out in an efficacious andrelatively simple manner.

A further object of the invention is to provide a process for producingcitric acid, isocitric acid and/or microbial cells by fermentation whichmay be carried out advantageously on an industrial scale at low cost togive a high yield of product.

A still further object of the invention is to provide citric acid,isocitric acid and/or microorganism cell bodres.

These and other objects and advantages of the present invention willbecome apparent to those skilled in the art from a consideration of thefollowing specification and claims.

In accordance with the present invention, it has been found that, whenattempts were made to produce microbial cells from hydrocarbons, aconsiderable amount of citric acid was accumulated in the culture liquorby adding an alcohol to the medium without affecting the productionyield of the microbial cells from the hydrocarbons. It was furtherdiscovered that the protein content of the thus-obtained microbial cellswas slightly increased, as compared with the case where the onlyhydrocarbons were used as the carbon source.

Other studies have shown that certain additives to the nutrient mediumhave a great effect upon the accumulation and yield of citric acid andisocitric acid in the culture liquor. Using appropriate additives, theisolation and recovery of the product citric acid or isocitric acid canbe made much simpler. In order to detect chemicals having an effect uponthe development or increase in the productivity of citric acid, using ahydrocarbon-assimilable microorganism and hydrocarbons as the maincarbon source, the present inventors have made careful studies 3 ofvarious aconitaseinhibiting agents, metal-chelating agents, alcohols,surfactants, deionizing agents, fluorides, etc., and have found thatpotassium ferrocyanide and monofluoroacetic acid have a remarkableeffect. Hence, it has been found that the yield of isocitric acid isgreatly increased by the addition of potassium ferrocyanide to themedium, whereas the yield of citric acid is considerably increased bythe addition of monofluoroacetic acid to the medium. The addition ofthese compounds to the media not only effects the yield and accumulationof isocitric acid and citric acid, but also has a considerable effectupon the variation in formation ratio of these organic acids by means ofthe increase in the productivities thereof.

Discussing this latter embodiment of the invention in more detail, theaddition of an effective amount up to about 5% by weight of potassiumferrocyanide to the medium provides the benefits discussed hereinabove.The amount of monofluoroacetic acid to be added to the medium rangesfrom an effective amount up to about mM. However, it is difficult todraw a strict line of concentration since the amounts will varydepending upon the particular microorganism employer and thefermentation conditions. Moreover, the concentration can be varied inrelation to the time of addition. Althoughthe additives can be providedto the nutrient medium all at one time or intermittently, the optimumtime for the addition is during the relatively initial stages ofculturing, with the best results being obtained usually between 0 and 24hours of cultivation.

The fact that the productivity of isocitric acid is enhanced by theaddition of potassium ferrocyanide to the medium is novel to the art andis believed to constitute an extremely advantageous way for producingcitric acid as well as isocitric acid, which are important metabolismintermediate products.

While the formation and accumulation of citric acid usingmonofluoroacetic acid as an additive in the medium has been reported inthe prior art, this procedure was conducted using a saccharine materialas the carbon source. This provision has not been conducted using ahydrocarbon-assimilable microorganism. The present invention is thefirst to disclose the addition of monofluoroacetic acid to a nutrientmedium to obtain considerable amounts of citric acid, making it possibleto conduct the process of the invention on an industrial scale.Moreover, the culturing procedure is greatly simplified in accordancewith the invention by the fact that the monofluoroacetic acid can beadded to the medium at the initiation of culturing.

Particularly when hydrocarbons are used as the carbon source, a smoothaction of aconitase activity is thought to be necessary, and, thus, itseems that the effect of potassium ferrocyanide or monofluoroacetic acidis so peculiar that it cannot be simply explained only from the functionwhich has been heretofore reported in the literature. Yet, using theseadditives, the present invention provides a procedure for producingcitric acid and isocitric acid which can be conducted commercially.

in connection with the first embodiment of the invention, wherein citricacid and microbial cells are produced by fermentation using an alcoholas the additive in the medium, it has been found that alcohols ingeneral may be used in this connection. However, alcohols having one to20 carbon atoms are particularly effective, and the preferred alcoholsto be employed are saturated alcohols having one to 20 carbon atoms.These alcohols themselves are effectively assimilated by themicroorganisms employed and also show an inhibiting action. Thus, it isnecessary to properly select the concentration thereof.

The effects of adding various alcohols upon the yield of microbialcells, the cell body protein content and the formation of citric acidare shown in Table l below, wherein microbial cells were produced fromnparaffins using Candida zeylanoides ATCC 15585. These experimental dataare the results obtained when culturing was carried out with aerobicshaking in flasks for 36 hours, the composition of the culturing mediumbeing as follows:

n-paraffin (C C C C (equal volume mixture): 2.0%(V/V) NH Cl: 2.0%(W/V)KH,P0,= 0.1 K HPO 0.1 MgSO 7H,O: 0.1 FeSO '7H O: 0.002 ZnSO '7H O: 0.004MnSO '4I-1,O 0.001 CuSOySl-LO: 0.001 Cornsteep liquor: 0.1 Thiaminehydrochloride: 3007/1 CaCO;,: 0.5%(W/V TABLE 1 Effects of addition ofvarious alcohols Crude Yield Alcohol Concent- Yield of protein of rationcell content citric %(V/V) bodies of cell acid (dry) bodies mg./ml.mg./ml. (dry) No addition 0 11.9 62.4 0

Methanol 0.1 12.1 62.4 0.5 0.2 12.3 62.7 0.8 0.3 12.6 63.1 1.5 0.4 12.762.9 2.6 0.5 13.1 63.2 3.4

Ethanol 0.2 14.6 62.8 0.4 0.4 17.2 62.6 0.6 0.6 19.1 63.3 1.2 0.8 21.463.9 2.0 1.0 23.2 64.5 2.5

Butanol 0.1 12.0 62.4 0.3 0.2 12.2 62.8 0.7 0.3 12.6 63.6 1.2 0.4 12.563.2 1.8 0.5 12.3 63.3 2.1

Lauryl 0.2 14.7 63.6 0.3 alcohol 0.4 15.6 64.2 0.7 0.6 17.7 65.1 1.6 0.821.6 66.0 2.8 1.0 24.7 65.7 3.6

Stearyl 0.2 13.1 63.1 0.3 alcohol 0.4 15.9 63.8 0.6 0.6 18.2 64.4 1.60.8 21.8 65.8 2.1 1.0 23.7 65.1 3.3

Oleyl 0.2 13.1 63.1 0.4 alcohol 0.4 16.1 65.2 0.8 0.6 19.2 66.0 1.9 0.821.1 65.3 2.6 1.0 24.3 65.7 3.8

As is clear from Table 1, no citric acid is formed at all in the cultureliquor when an alcohol is not added to the medium. However, a smallamount of citric acid is formed and accumulated when various alcoholsare added thereto. Furthermore, the microorganism employed is capable ofassimilating alcohols having 10 to 20 carbon atoms and forms microbialcells in a yield (based on carbon) which is almost as good as that fromn-paraffins or ethanol. in addition, as is clear from Table 1, it isnoted that the protein content is somewhat increased by the addition ofan alcohol to the medium.

Hydrocarbon-assimilating microorganisms, including bacteria, yeasts,molds, etc. in a wide range, can be employed in the present invention.Preferred bacteria are those belonging to the genera Arthrobacter,Corynebacterium, Brevibacterium or Nocardia. The preferred yeasts arethose belonging to the genera Candida, Torulopsis, Endomyces, Pichia,l-lansenula, Mycoderma or Endomycopsis. Preferred molds are those of thegenus Aspergillus or the genus Penicillium.

Either a synthetic culture medium or a natural nutrient medium issuitable in the fermentation process of the present invention as long asit contains the essential nutrients for the growth of the microorganismstrain employed. Such nutrients are well known in the art and includesubstances such as a carbon source, a nitrogen source, inorganiccompounds and the like which are utilized by the microorganism employedin appropriate amounts. The fermentation in connection with the presentinvention is conducted in an aqueous nutrient medium containing ahydrocarbon or a mixture of hydrocarbons as the main carbon source. Suchhydrocarbons include straight and branched-chain paraffins (alkanes),particularly those having from 1 l to 20 carbon atoms, such asn-pentane, n-octane, n-decane, ndodecane, n-hexadecane, isopentane,osooctane, etc., cycloparaffins such as cyclohexane and cyclooctane,straightand branched-chain olefins such as pentene-2, hexene-l,octene-l, octene-2, etc., cycloolefins such as cyclohexene, aromatichydrocarbons such as benzene, o-xylene, etc., and mixtures thereof andmixed hydrocarbons such as kerosene, light oils, heavy oils, paraffinoils, etc. Small amounts of other carbon sources such as carbohydrates,for example, glucose, fructose, maltose, sucrose, starch, starchhydrolysate, molasses, etc., or any other suitable carbon source such asglycerol, mannitol, sorbitol, organic acids, etc. may be used in thefermentation medium along with the hydrocarbon. These substances may beused either singly or in mixtures of two or more.

As a nitrogen source, various kinds of inorganic or organic salts orcompounds, such as urea or aqueous ammonia or ammonium salts such asammonium chloride, ammonium sulfate, ammonium nitrate, ammoniumphosphate, ammonium acetate, etc., or one or more than one amino acidmixed in combination, or natural substances containing nitrogen, such ascornsteep liquor, yeast extract, meat extract, fish meal, peptone,bouillon, casein hydrolysates, fish solubles, rice bran extract, etc.may be employed. These substances may also be used either singly or incombinations of two or more.

Inorganic compounds which may be added to the culture medium includemagnesium sulfate, sodium phosphate, potassium dihydrogen phosphate,potassium monohydrogen phosphate, iron sulfate or other iron salts,manganese chloride, calcium chloride; sodium chloride, etc. Moreover, itmay also be necessary to add certain essential nutrients to the culturemedium, depending upon the particular microorganism employed, such asamino acids, for example, aspartic acid, threonine, methionine, etc.,and/or vitamins, for example, biotin, thiamine, cobalamin and the like.

In some instances, it may be necessary or desirable to use an alcohol, amonoor dicarboxylic acid or a natural fat or oil, or a mixture thereof,as the carbon source or at least a part of the carbon source in themedium. The addition of vegetable oil, an animal oil and/or a surfactantmay also be effective in helping to increase the yield of product.

The fermentation or culturing of the microorganisms is conducted underaerobic conditions, such as aerobic shaking of the culture or withstirring and aeration of a submerged culture, at a temperature of, forexample, about to 45 C. and at a pH of, for example, about 1.0 to 9.0 inthe case of the former embodiment wherein an alcohol is added to themedium, and at a temperature of, for example, about 20 to 37 C. and at apH of, for example, about 1.0 to 7.5 in the case of the latterembodiment wherein potassium ferrocyanide or monofluoroacetic acid isadded to the medium.

The following examples are given merely as illustrative of the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the percentages therein and throughout the application are byweight per volume of water. The desired products are recovered byconventional means, such as ion exchange resin treatment, extractionwith solvents, precipitation, adsorption, chromatography or the like.

EXAMPLE 1 18.9 liters of a fermentation medium having-the followingcomposition is placed into a 30-liter volume jar fermentor:

2.5% (V/V) n-paraffins (C C C, )(equal volume mixture) 2.0% (W/V) NH CI0.1% KH PO 0.1% K HPO 0.002% FeSO '7H O 0.001% ZnSO -7H O 0.0005% CuSO'5H- O 0.0002% H BO 500y/ml Nonion LP-20R(surfactant) 1 ml/l soybean oil200y/l thiamine hydrochloride 0.05% cornsteep liquor One liter of amicrobial seed liquor of Torulopsir famata ATCC 15586 is added to theabove fermentation medium. Then, 0.1 liter of ethanol is also addedthereto. Culturing is carried out at a cultivation temperature of 34 C.with aeration at the rate of 20 liters/- minute and with stirring at therate of 500 r.p.m. The pH is kept at 6.50 with 18% aqueous ammoniaduring the culturing.

After 42 hours, the culturing is completed, and the concentration ofmicrobial cells in the culture liquor reaches 20 mg/ml. The yield ofcitric acid is 3.0 mg/ml.

As a comparison, culturing is carried out in the same manner in a mediumcontaining no ethanol. The concentration of formed cell bodies is 14mg/ml, and no by-product citric acid is observed at all.

The microbial cells obtained as a result of adding ethanol to the mediumare repeatedly washed several times in a Sharples centrifugal separatorand subjected to centrifugal separation. After freeze-drying, 365 g. ofcell bodies is obtained. The protein content per cell body is 61%.However, when cultivation is conducted in a medium containing noethanol, only 2.46 g. of cell bodies is obtained with a protein contentof 59%.

After removing the microbial cells'from the culture liquor, 200 g. ofcalcium chloride is added, and the pH is adjusted to 8.0 with aqueousammonia. The resultant EXAMPLE 2 Three hundred ml. of a microbial seedsolution of Nocardia paraffinica ATCC 21 198 is added to 3 liters of afermentation medium contained in a 5-liter jar fermentor and having thefollowing composition:

3.5% (V/V) n-paraffins (C C C (equal volume mixture) 0.1% (W/V) KH PO0.05% cornsteep liquors 2.5% (NH ),SO

0.01% CaCl,

Added to the above fermentation medium is 15 ml. of methanol.Cultivation is then carried out at 28 C. with aeration at the rate of 3liters/minute and with stirring at the rate of 650 r.p.m. The pH is keptat 7.0 with 18% aqueous ammonia during the cultivation.

Culturing is finished after 40 hours. The concentration of formed cellbodies is 21.0 mg/ml, and the yield of citric acid is 2.0 mg/ml. Whenculturing is conducted without the addition of methanol, theconcentration of formed cell bodies is 20.8 mg/ml but the formation ofcitric acid is not observed at all.

By subjecting the culture liquor to the same procedures as described inExample 1, microbial cells and crystals of calcium citrate are obtained.The yield amounts to 53 g. of cell bodies (dry) and 6.1 g. of calciumcitrate. When culturing in the medium containing no methanol, 52 g. ofcell bodies (dry) is obtained. The protein content of the microbialcells obtained in accordance with the invention is 57.1%, while theprotein content of the microbial cells obtained by culturing in themedium containing no methanol is 56.3%.

EXAMPLE 3 Three liters ofa fermentation medium having the followingcomposition is placed in a 5-liter jar fermentor:

4% (V/V) n-paraffins (C C C (equal volume mixture) 2.0% (W/V) NH NO 0.1%Kl-l Po,

0.05% K HPO,

0.002% FeSO -7H,O

0.0005% CuSO -5H,O

200y/m1 Nonion OT-22l (surfactant) 1 ml/l soybean oil 1 mg/l thiaminehydrochloride 0.5% (V/V) lauryl alcohol Then, 300 ml. ofa culturesolution of Penicillium janthinellum ATCC 13154 is added thereto as theseed microorganism. Culturing is carried out at 26 C. with aeration atthe rate of 3 liters/minute and with stirring at 650 r.p.m. for 3 days.The initial pH is 6.0.

The concentration of formed cell bodies reaches 30 mg/ml, together witha yield of citric acid of 1.8 mg/ml. When culturing is conducted in amedium containing no lauryl alcohol, the concentration of formed cellbodies is 24 mg/ml, and no citric acid is formed at all. By

65 sub ectingthe culture liquor to the same treatment as described inExample 1, 85 g. of cell bodies and 5.6 g. of crystals of calciumcitrate are obtained. The protein content of the cell bodies is 36%.When cultivation is conducted in a medium containing no lauryl alcohol,69 g. of cell bodies is obtained having a protein content of 31%.

EXAMPLE 4 Placed in a 5-liter jar fermentor is 3 liters of afermentation medium comprising:

3.0% (V/V) n-paraffins (C C C (equal volume mixture) 0.1% (V/V) KHJO,

0.1% K,HPO

0.3% KCl 0.005% FeSO -7H,O

0.001% MnSO -4H,O

0.02% cornsteep liquor 2.5% (NH ),SO

0.3% CaCl,

100 'y/l thiamine hydrochloride Added to the above medium is 300 ml. ofamicrobial seed solution of Arthrobacter paraffineus ATCC 15591.Culturing is then carried out at 30C. with aeration at the rate of 3liters/minute and with stirring at the rate of 800 r.p.m., while keepingthe pH at 6.80 with 28% aqueous ammonia. Then, 6 hours after theinitiation of culturing, 15 ml. of n-amyl alcohol is added thereto. Theyield of microbial cells reaches a maxithereof being 18.5 mg/ml. Theyield of citric acid is 1.3 mg/ml. Culturing conducted as a control inthe same medium but containing no n-amyl alcohol gives a concentrationof microbial cells of 18.0 mg/ml and no formation of citric acid at all.

By subjecting the culture liquor obtained by adding n-amyl alcohol tothe medium to the same treatment as described in Example 1, 49 g. ofmicrobial cells (dry) and 4.5 g. of calcium citrate are obtained. Theprotein content of the microbial cells is 51.0%. From the controlculture liquor obtained by cultivation without namyl alcohol, 48 g. ofmicrobial cells is obtained with a protein content of 48.2%.

EXAMPLE 5 Arthrobacter paraffineus ATCC 15591 is cultured at 30C. for 24hours with aerobic shaking in 20 ml. of a medium containing 0.25% yeastextract, 0.5% meat extract, 0.5% peptone and 0.25% sodium chloride at apH of 7.0 in a 250-ml. conical flask. The resulting culture istransferred, in a proportion of 10%, to 20 ml. of a fermentation mediumcontained in 500-ml. Sakaguchi flasks and having the followingcomposition:

0.1% Na HPO JZI-LO 0.01% FeSOfll-LO 0.002% MnSO -4H,O

0.05% cornsteep liquor 2.0% NH NO 5% n-paraffin mixture (equal volumemixture of C C13 and C14) 5% CaCO,

Culturing is conducted at 30C. with reciprocating shaking. Sodiummonofluoroacetate is added thereto 6 hours after the initiation ofculturing in a concentration of 0.1%. Then, the cultivation is continuedfor 4 days,

mum after 24 hours of culturing, the concentration whereby 18.2 mg/ml ofcalcium citrate (in terms of citric acid) is found to be accumulated inthe culture liquor. Using the same medium in a control experiment, only9.3 mg/ml of citric acid is obtained when no sodium monofluoroacetate isadded to the medium.

An equal volume of 2 N-HCl is added to the culture liquor after thecompletion of culturing in order to dissolve all of the calcium citrate,and the microbial cells are removed therefrom by centrifugation. NH OHis added to the resulting supernatant liquor to adjust the pH to 8.0,and after heating, about 360 mg. of the resulting calcium citrate isseparated by filtration. By repeating this treatment once more, 332 mg.of calcium citrate crystals is obtained.

EXAMPLE 6 Candida zeylanoides ATCC 15585 is cultured at 30C. for 2 dayswith aerobic shaking in a Sakaguchi flask containing 20 ml. of a mediumcomprising a n-paraffin mixture (V/V), 0.5% meat extract, 0.5%polypeptone, 0.25% sodium chloride and 5% CaCO at a pH of 6.0. Theresulting culture is transferred, in a proportion of l0%, to Sakaguchiflasks, each containing 20 ml. of a fermentation medium comprising:

% n-paraffin mixture (V/V) 0.1% KH PO 0.05% MgSO -7H O 50,0 y/l MnSO -4HO 500 'y/l CuSO -5H O 200 mg/l Nonion OT-22l (surfactant) 5 ml/l soybeanoil l mg/l thiamine 0.01% sodium monofluoroacetate 8% CaCO The pH of theabove medium is 6.0.

Culturing is carried out for 4 days under the same conditions asdescribed in Example 5. After the completion of culturing, 105 mg/ml ofcalcium citrate (in terms of citric acid) and 12 mg/ml of calciumisocitrate (in terms of isocitric acid) are found to be accumulated inthe resultant culture liquor. in a control experiment, culturing iscarried out in the same manner but in a medium free of themonofluoroacetic acid. In this case, only 65 mg/ml of calcium citrate(in terms of citric acid) is formed, while 63 mg/ml of calciumisocitrate (in terms of isocitric acid) is accumulated in the cultureliquor.

By treating the culture liquor after the completion of cultivation inthe same manner as described in Example 5, 2.05 g. of calcium citrate isobtained from the medium cultivated in accordance with the presentinvention. The crystals obtained contain 10.3% of calcium isocitrate (interms of isocitric acid).

EXAMPLE 7 Candida lipolytica ATCC 866] is cultured at 30C. for 2 dayswith aerobic shaking in a Sakaguchi flask containing 20 ml. of a mediumcomprising a 5% nparaffin mixture (V/V), 0.5% meat extract, 0.5% polypeptone, 0.25% sodium chloride and 5% CaCO at a pH of 6.0. The resultingculture is transferred, in a pro- 6 10% n-paraffin mixture (V/V) 0.3% NHNO 0.1% KH PO 500 11/1 MnSO -4H O 500 'y/l CuSO -5H O 200 'y/l H 200mg/l Nonion OT-22l (surfactant) 5 ml/l soybean oil 0.1% cornsteep liquor8% CaCO The pH of this medium is 6.0.

Culturing is carried out with aerobic shaking under the same conditionsas described in Example 5, and potassium ferrocyanide is added to themedium 24 hours after the initiation of culturing in order to give aconcentration of 2.0% by weight therein. Culturing is then continued fora further 72 hours and, as a result, 4.6 mg/ml of calcium citrate (interms of citric acid) and 10.8 mg/ml of calcium isocitrate (in terms ofisocitric acid) are found to be accumulated in the resultant cultureliquor.

The obtained culture liquor is collected from 50 flasks subjected tocultivation as described above, and H SO is added thereto. The cultureliquor is then filtered in order to remove the calcium salts and themicroorganisms therefrom. Then, saturated Ba(OH) is added to the cultureliquor, wereby Ba salts are obtained. After the steps of lactonation,methylation, demethylation and lactonation, 1.25 g. of the lactone ofisocitric acid is obtained.

When culturing is carried out in a control experiment in the same mannerbut in a medium containing no potassium ferrocyanide, 7.5 mg/ml ofcalcium citrate (in terms of citric acid) and 7.4 mg/ml of calciumisocitrate (in terms of isocitric acid) are accumulated in thefermentation liquor.

EXAMPLE 8 Culturing is conducted in the same manner as described inExample 7, using Candida zeylanoides ATCC 15585 as the microorganism. Asa result, 12.1 mg/ml of calcium isocitrate (in terms of isocitric acid)and 5.0 mg/ml of calcium citrate (in terms of citric acid) are formed inthe culture liquor at the completion of cultivation. By following theprocedure described in Example 7, 1.44 g. of the lactone of isocitricacid is obtained.

When cultivation is carried out in the same manner but in a mediumcontaining no potassium ferrocyanide as a control, 7.3 mg/ml of calciumisocitrate (in terms of isocitric acid) and 7.5 mg/ml of calcium citrate(in terms of citric acid) are formed in the culture liquor at thecompletion of culturing.

EXAMPLE 9 Penicillium janthinellum 581 ATCC 13154 is cultured at 28C. ona malt extract agar-agar slant medium for 3 days. One platinum loop ofthe resultant seed culture is inoculated onto 20 ml. of a seed culturemedium contained in a 250-ml. flask and having the following composition(pH 6.0):

5% n-paraffin mixture (C C C (V/V) 0.025% KH PO 0.001% CaCl,-2H,O

0.001% MnSO '4H O mg/l FeSO '7H- O 0.6 mg/l H 80 0.3% cornsteep liquor2% soybean oil 3% CaCO Culturing is carried out with aerobic shaking ofthe culture at 28C. for 3 days, and 2 ml. of the resulting cultureliquor is transferred to 20 ml. of a fermentation medium having the samecomposition [except for the 10% n-paraffin mixture (V/V) and 5% CaCOcontained in a Sakaguchi flask. Then, culturing is carried out withaerobic shaking at the rate of 130 reciprocations/minute at 30C. After24 hours of cultivation, sodium monofluoroacetate is added to the mediumin a concentration of 0.01%. Culturing is continued for a further 5 daysand, as a result, 30 mg/ml of calcium citrate (in terms of citric acid)is formed in the culture liquor after the completion of culturing.

In a control experiment, where no sodium monofluoroacetate is added tothe medium, only 12 mg/ml of calcium citrate (in terms of citric acid)is found to be accumulated in the resultant culture liquor.

From the above description, it can be seen that an alkali metalferrocyanide, such as sodium ferrocyanide or potassium ferrocyanide, canbe employed as the additive in obtaining increased yields of isocitircacid. In a similar manner, the addition of monofluoroacetic acid or thealkali metal salts thereof, such as sodium monofluoroacetate orpotassium monofluoroacetate, is effective in increasing the yield ofcitric acid.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included herein.

We claim:

1. A process for producing citric acid together with microbial cells byfermentation which comprises culturing a hydrocarbon-assimilatingmicroorganism capable of producing citric acid under aerobic conditionsin an aqueous nutrient medium containing at least one hydrocarbon as themain carbon source in the presence of at least one alcohol withoutcontrol of said microbial growth, said nutrient medium being able tosupport microbial growth but being unable to support production ofcitric acid substantially in the absence of said alcohol without controlof microbial growth, accumulating citric acid and the resultingmicrobial cells in the culture liquor, and recovering the citric acidand the microbial cells therefrom.

2. The process of claim 1, wherein said microorganism belongs to a genusselected from the group consisting of Arthrobacter, Corynebacterium,Brevibacterium, Nocardia, Candida, Torulopsis, Endomyces, Pichia,Hansenula, Mycoderma, Endomycopsis, Aspergillus and Penicillium.

3. The process of claim 2, wherein culturing is carried out at atemperature of about 15 to 45C. and at a pH of about 1.0 to 9.0.

4. The process of claim 1, wherein said alcohol contains from one to 20carbon atoms.

5. The process of claim 1, wherein said alcohol is an saturated alcoholhaving from one to 20 carbon atoms.

6. The process of claim 1, wherein approximately 0.1 to 1.0% (V/V) ofsaid alcohol is added to the medium.

7. The process of claim 1, wherein said hydrocarbon is an n-paraffinhaving from 11 to 20 carbon atoms.

8. The process of claim 1, wherein the nutrient medium further includesa surfactant.

9. The process of claim 1, wherein said microorganism is selected fromthe group consisting of Torulopsis famata, Nocardia paraffinica,Penicillium janthinellum and Arthrobacter paraffineus.

10. The process of claim 1, wherein said nutrient medium containssufficient nitrogen source to avoid adverse affect upon the productionof said microbial cells.

11. A process of producing citric acid together with microbial cells byfermentation which comprises culturing a hydrocarbon-assimilatingmicroorganism capable of producing citric acid and belonging to a genusselected from the group consisting of Arthrobacter, Corynebacterium,Brevibacterium, Nocardia, Candida, Torulopsis, Endomyces, Pichia,Hansenula, Mycoderma, Endomycopsis, Aspergillus and Penicillium underaerobic conditions without control of microbial growth at a temperatureof about 15 to 45C. and a pH of about 1.0 to 9.0 in an aqueous nutrientmedium containing at least one hydrocarbon as the main carbon source inthe presence of approximately 0.1 to 1.0% by weight of at least onesaturated alcohol having from one to 20 carbon atoms, said nutrientmedium being able to support production of microbial cells, but beingunable to support production of citric acid substantially in the absenceof said alcohol without control of microbial growth, accumulating citricacid and the microbial cells in the culture liquor, and recovering thecitric acid and the microbial cells therefrom.

12. The process of claim 11, wherein said hydrocarbon is an n-paraffinhaving from 1 l to 20 carbon atoms.

13. The process of claim 11, wherein said microor-' ganism is selectedfrom the group consisting of Torulapsis famata, Nocardia paraffinica,Penicilluum janthinellum and Arthrobacter paraffineus.

14. The process of claim 11, wherein said nutrient medium containssufficient nitrogen source to avoid adverse affect on the production ofsaid microbial cells. l t I I

2. The process of claim 1, wherein said microorganism belongs to a genusselected from the group consisting of Arthrobacter, Corynebacterium,Brevibacterium, Nocardia, Candida, Torulopsis, Endomyces, Pichia,Hansenula, Mycoderma, Endomycopsis, Aspergillus and Penicillium.
 3. Theprocess of claim 2, wherein culturing is carried out at a temperature ofabout 15* to 45*C. and at a pH of about 1.0 to 9.0.
 4. The process ofclaim 1, wherein said alcohol contains from one to 20 carbon atoms. 5.The process of claim 1, wherein said alcohol is an saturated alcoholhaving from one to 20 carbon atoms.
 6. The process of claim 1, whereinapproximately 0.1 to 1.0% (V/V) of said alcohol is added to the medium.7. The process of claim 1, wherein said hydrocarbon is an n-paraffinhaving from 11 to 20 carbon atoms.
 8. The process of claim 1, whereinthe nutrient medium further includes a surfactant.
 9. The process ofclaim 1, wherein said microorganism is selected from the groupconsisting of TorulopSis famata, Nocardia paraffinica, Penicilliumjanthinellum and Arthrobacter paraffineus.
 10. The process of claim 1,wherein said nutrient medium contains sufficient nitrogen source toavoid adverse affect upon the production of said microbial cells.
 11. Aprocess of producing citric acid together with microbial cells byfermentation which comprises culturing a hydrocarbon-assimilatingmicroorganism capable of producing citric acid and belonging to a genusselected from the group consisting of Arthrobacter, Corynebacterium,Brevibacterium, Nocardia, Candida, Torulopsis, Endomyces, Pichia,Hansenula, Mycoderma, Endomycopsis, Aspergillus and Penicillium underaerobic conditions without control of microbial growth at a temperatureof about 15* to 45*C. and a pH of about 1.0 to 9.0 in an aqueousnutrient medium containing at least one hydrocarbon as the main carbonsource in the presence of approximately 0.1 to 1.0% by weight of atleast one saturated alcohol having from one to 20 carbon atoms, saidnutrient medium being able to support production of microbial cells, butbeing unable to support production of citric acid substantially in theabsence of said alcohol without control of microbial growth,accumulating citric acid and the microbial cells in the culture liquor,and recovering the citric acid and the microbial cells therefrom. 12.The process of claim 11, wherein said hydrocarbon is an n-paraffinhaving from 11 to 20 carbon atoms.
 13. The process of claim 11, whereinsaid microorganism is selected from the group consisting of Torulapsisfamata, Nocardia paraffinica, Penicilluum janthinellum and Arthrobacterparaffineus.
 14. The process of claim 11, wherein said nutrient mediumcontains sufficient nitrogen source to avoid adverse affect on theproduction of said microbial cells.